It has been unclear to which antimicrobial activities (e.g., anti-gram-positive bacterial, anti-gram-negative bacterial, antifungal, antiparasitic, and antiviral activities) of antimicrobial peptides (AMPs) a given physiochemical property matters most. This is the first computational study using large-scale AMPs to examine the relationships between antimicrobial activities and two major physiochemical properties of AMPs—amphipathicity and net charge. The results showed that among all kinds of antimicrobial activities, amphipathicity and net charge best differentiated between AMPs with and without anti-gram-negative bacterial activities. In terms of amphipathicity and charge, all the AMPs whose activities were significantly associated with amphipathicity and net charge were alike except those with anti-gram-positive bacterial activities. Furthermore, the higher the amphipathic value, the greater the proportion of AMPs possessing both antibacterial and antifungal activities. This dose–response-like pattern suggests a possible causal relationship—dual antibacterial and antifungal activities of AMPs may be attributable to amphipathicity. These novel findings could be useful for identifying potent AMPs computationally.